U.S. patent application number 11/615578 was filed with the patent office on 2007-07-12 for anthrax and small pox replikins and methods of use.
Invention is credited to Elenore S. Bogoch, Samuel Bogoch.
Application Number | 20070160624 11/615578 |
Document ID | / |
Family ID | 38232963 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070160624 |
Kind Code |
A1 |
Bogoch; Samuel ; et
al. |
July 12, 2007 |
ANTHRAX AND SMALL POX REPLIKINS AND METHODS OF USE
Abstract
Isolated peptides of the Bacillus anthracis Anthrax Toxin Lethal
factor Protein pX01-107, antibodies specific for the peptides and
methods of stimulating the immune response of a subject to produce
antibodies to the Bacillus anthracis Anthrax Toxin Lethal factor
Protein pX01-107 are disclosed. Also disclosed are isolated
peptides of the Small Pox Virus Surface Antigen S Precursor
Protein, antibodies specific for the peptides and methods of
stimulating the immune response of a subject to produce antibodies
to the Small Pox Virus Surface Antigen S Precursor Protein.
Inventors: |
Bogoch; Samuel; (New York,
NY) ; Bogoch; Elenore S.; (New York, NY) |
Correspondence
Address: |
KENYON & KENYON LLP
1500 K STREET N.W.
SUITE 700
WASHINGTON
DC
20005
US
|
Family ID: |
38232963 |
Appl. No.: |
11/615578 |
Filed: |
December 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09984056 |
Oct 26, 2001 |
7176275 |
|
|
11615578 |
Dec 22, 2006 |
|
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60303396 |
Jul 9, 2001 |
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60278761 |
Mar 27, 2001 |
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Current U.S.
Class: |
424/190.1 ;
514/44R; 530/324; 530/325; 530/327; 530/328; 530/329; 530/388.3;
530/388.4; 536/23.1 |
Current CPC
Class: |
C12N 2710/24122
20130101; A61K 39/00 20130101; A61P 31/04 20180101; C07K 14/005
20130101; C07K 16/081 20130101 |
Class at
Publication: |
424/190.1 ;
530/388.4; 530/324; 530/325; 530/327; 530/328; 530/329; 530/388.3;
514/044; 536/023.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/02 20060101 A61K039/02; C07H 21/02 20060101
C07H021/02; C07K 14/32 20060101 C07K014/32; C07K 14/07 20060101
C07K014/07; C07K 16/08 20060101 C07K016/08; C07K 16/12 20060101
C07K016/12 |
Claims
1-9. (canceled)
10. An isolated small pox virus peptide consisting of 7 to about 50
amino acids wherein said peptide is isolated by identifying a motif
consisting of (1) at least one lysine residue located at a first
terminus of said motif and at least one lysine residue or at least
one histidine residue located at a second terminus of said motif;
(2) a first lysine residue located six to ten residues from a
second lysine residue; (3) at least one histidine residue; and (4)
at least 6% lysine residues, selecting said identified motif and
isolating said replikin peptide comprising said identified
motif.
11. The peptide of claim 10 comprising the amino acid sequence as
set forth in SEQ ID NO. 99.
12. The peptide of claim 10 comprising the amino acid sequence as
set forth in SEQ ID NO. 100.
13. The peptide of claim 10 comprising the amino acid sequence as
set forth in SEQ ID NO. 101.
14. The peptide of claim 10 comprising the amino acid sequence as
set forth in SEQ ID NO. 102.
15. The peptide of claim 10 comprising the amino acid sequence as
set forth in SEQ ID NO. 103.
16-21. (canceled)
22. A method of stimulating the immune system of a subject to
produce antibodies to small pox virus comprising administering an
effective amount of at least one small pox virus peptide of claim
10.
23. The method of claim 22 wherein the at least one peptide is
selected from the group consisting of SEQ ID NO. 99, SEQ ID NO.
100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103 and
combinations thereof.
24-27. (canceled)
28. The isolated small pox virus peptide of claim 10 selected from
the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102 and SEQ ID NO:103.
29. The isolated small pox virus peptide of claim 28 consisting of
SEQ ID NO:99.
30. The isolated small pox virus peptide of claim 28 consisting of
SEQ ID NO:100.
31. The isolated small pox virus peptide of claim 28 consisting of
SEQ ID NO:101.
32. The isolated small pox virus peptide of claim 28 consisting of
SEQ ID NO: 102.
33. The isolated small pox virus peptide of claim 28 consisting of
SEQ ID NO: 103.
34. An isolated nucleic acid sequence encoding the small pox virus
peptide of claim 10.
35. The isolated nucleic acid sequence of claim 34 wherein said
small pox virus peptide is selected from the group consisting of
SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102 and SEQ
ID NO:103.
36. The isolated nucleic acid sequence of claim 35 consisting of
the nucleic acid sequence encoding SEQ ID NO:99.
37. The isolated nucleic acid sequence of claim 35 consisting of
the nucleic acid sequence encoding SEQ ID NO:100.
38. The isolated nucleic acid sequence of claim 35 consisting of
the nucleic acid sequence encoding SEQ ID NO:101.
39. The isolated nucleic acid sequence of claim 35 consisting of
the nucleic acid sequence encoding SEQ ID NO:102.
40. The isolated nucleic acid sequence of claim 35 consisting of
the nucleic acid sequence encoding SEQ ID NO:103.
Description
CROSS REFERENCE TO OTHER APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/984,056, filed Oct. 26, 2001 and claims priority to Provisional
Application Ser. No. 60/303,396 filed Jul. 9, 2001 and 60/278,761
filed Mar. 27, 2001, which are incorporated herein in their
entirety by reference thereto.
FIELD OF THE INVENTION
[0002] This invention relates to the identification and use of
Replikins, a class of peptides that share structural
characteristics. In particular, this invention relates to Replikins
which have been identified in Bacillus anthracis and Small Pox
Virus (Variola).
BACKGROUND OF THE INVENTION
[0003] Glycoprotein 10B is a membrane glycoprotein isolated from
brain glioblastoma multiforme, lymphoma and breast cancer cells
(U.S. Pat. No. 6,242,578 B1). A constituent peptide of Aglyco 1OB,
malignin, is enriched in cell membranes tenfold during anaerobic
replication. Hydrolysis and mass spectrometry of malignin yielded a
16-mer peptide including (SEQ ID NO.: 1) kagvaflhkk. This peptide,
which is absent from the normal human genome, was assumed to be
acquired.
SUMMARY OF THE INVENTION
[0004] In one aspect of the invention there are provided isolated
Bacillus anthracis (Anthrax) peptides containing a replikin
sequence. The Anthrax peptides comprise from 7 to about 50 amino
acids including (1) at least one lysine residue located six to ten
amino acid residues from a second lysine residue; (2) at least one
histidine residue; and (3) at least 6% lysine residues. In another
embodiment of this aspect of the invention there are provided Small
Pox Virus peptides containing a replikin sequence which comprises
from 7 to about 50 amino acids including (1) at least one lysine
residue located six to ten amino acid residues from a second lysine
residue; (2) at least one histidine residue; and (3) at least 6%
lysine residues.
[0005] In another aspect of the invention there is provided a
process for stimulating the immune system of a subject to produce
antibodies that bind specifically to Anthrax polypeptides
containing a replikin sequence, said process comprising
administering to the subject an effective amount of a dosage of a
composition comprising at least one Anthrax replikin peptide. In a
preferred embodiment the composition comprises at least one peptide
selected from SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID
NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 79, SEQ ID NO. 98
or a combination thereof.
[0006] In another embodiment of this aspect of the invention there
is provided a process for stimulating the immune system of a
subject to produce antibodies that bind specifically to Small Pox
Virus polypeptides containing a replikin sequence, said process
comprising administering to the subject an effective amount of a
dosage of a composition comprising at least one Small Pox Virus
replikin peptide. In a preferred embodiment the composition
comprises a peptide selected from SEQ ID NO. 99, SEQ ID NO. 100,
SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, or a combination
thereof.
[0007] In another aspect of the invention there are provided
antisense nucleic acid molecules complementary to the coding strand
of the gene or to the mRNA encoding the Bacillus anthracis Anthrax
Lethal Factor Protein pX01-107 peptide, wherein said antisense
nucleic acid molecule is complementary to a nucleotide sequence
encoding the peptide of SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO.
93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ
ID NO. 98.
[0008] There are also provided antisense nucleic acid molecule
complementary to the coding strand of the gene or to the mRNA
encoding the Small Pox Virus Surface Antigen S Precursor Protein,
wherein said antisense nucleic acid molecule is complementary to a
nucleotide sequence encoding the peptide of SEQ ID NO. 99, SEQ ID
NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, or SEQ ID NO. 103.
[0009] As used herein, the term "peptide" refers to a compound of
two or more amino acids in which the carboxyl group of one is
united with an amino group of another, forming a peptide bond. The
term peptide is also used to denote the amino acid sequence
encoding such a compound. Thus, a peptide sequence may be a
subsequence of a larger polypeptide sequence. As used herein, a
Replikin peptide is peptide consisting essentially of 7 to about 50
amino acid including (1) at least one lysine residue located six to
ten amino acid residues from a second lysine residue; (2) at least
one histidine residue; and (3) at least 6% lysine residues.
Similarly, a replikin sequence is the amino acid sequence encoding
such a peptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a bar graph depicting the frequency of occurrence
of replikins in various protein groups.
[0011] FIG. 2 is a graph depicting the percentage of malignin per
milligram total membrane protein during anaerobic replication of
glioblastoma cells.
[0012] FIG. 3 is a bar graph showing amount of antimalignin
antibody produced in response to exposure to the recognin
16-mer.
[0013] FIG. 4A is a photograph of a blood smear taken with ordinary
and fluorescent light. FIG. 4B is a photograph of a blood smear
taken with and fluorescent light illustrating the presence of two
leukemic cells. FIG. 4C is a photograph of a dense layer of glioma
cells in the presence of antimalignin antibody. FIG. 4D and FIG. 4E
are photographs of the layer of cells in FIG. 4C taken at 30 and 45
minutes following addition of antimalignin antibody.
[0014] FIG. 4F is a bar graph showing the inhibition of growth of
small cell lung carcinoma cells in vitro by antimalignin
antibody.
[0015] FIG. 5 is a plot of the amount of antimalignin antibody
present in the serum of patients with benign or malignant breast
disease pre-and post surgery.
[0016] FIG. 6 is a box diagram depicting an embodiment of the
invention wherein a computer is used to carry out the
3-point-recognition method of identifying replikin and recognin
sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In one aspect of the invention there is provided a method
for identifying nucleotide or amino acid sequences that include a
recognin or replikin sequence. The method is referred to herein as
a 3-point-recognition method. By use of the "3-point recognition"
method, described herein below, a new class of peptides was
revealed in algae, yeast, fungi, amoebae, bacteria, plant and virus
proteins having replication, transformation, or redox functions.
This class of peptides is referred to herein as replikins.
[0018] One example of a replikin that was identified by the
3-point-recognition method, is the amino acid sequence, (SEQ ID
NO.: 2) hsikrelgiifdk, which occurs in Saccharomyces cerevisiae
"replication binding protein". Five replikins were found in amino
acids 1-163 of the "replicating protein" of tomato leaf curl Gemini
vinis. Amino acids 1-160 of this tomato virus protein bind DNA.
Another replikin, (SEQ ID NO.: 3) hkqkivapvk, is highly conserved
in 236 isolates of foot and mouth disease virus.
[0019] Although replikins were found to be present in only 1.5% of
published sequences identified by the PubMed data bank as "virus
peptides" as a whole, and in only 8.5% of sequences identified as
"brain peptides" plus "neuropeptides", surprisingly, replikins were
found in 100% of "tumor viruses", in 85% of "transforming
proteins," and 97% of "cancer proteins" (as categorized in the
PubMed data bank). The recognin, (SEQ ID NO.: 4) ykagvaflhkkndide,
was not found in published sequences of the human genome.
[0020] The 16-mer recognin peptide, (SEQ ID NO.: 4)
ykagvaflhkkndide, when synthesized and injected as vaccine into a
mammal, has been shown to produce antimalignin antibody, which is
cytotoxic to malignant replicating cells of several types at
picogram per cell amounts. Replikins identified in organisms such
as diatom plankton, H. pylori, tomato leaf curl virus, foot and
mouth disease virus, hepatitis B and C viruses, and HIV, also are
thus targets for diagnosis and treatment or as vaccines for the
control of replication of their respective virus source.
[0021] Table 1 illustrates the sequence of the malignin peptide,
the 16-mer recognin sequence, (SEQ ID NO.: 4) ykagvaflhkkndide.
TABLE-US-00001 TABLE 1 16-mer peptide sequence ykagvaflhkkndide
obtained from malignin by hydrolysis and mass spectrometry Method
By Which Fragment Obtained Auto-hydrolysis of malignin
Auto-hydrolysis immobilized Seq Fragment MH+ of malignin on
bromoacetyl Microwaved Microwaved ID NO. Identified (mass) Sequence
free in solution cellulose 5 seconds 30 seconds 19 1-3 381.21 (
)yka(g) + 20 1-5 537.30 ( )ykagv(a) + 21 2-6 445.28 (y)kagva(f) +
22 2-7 592.35 (Y)kagvaf(l) + 23 4-11 899.55 (a)gvaflhkk(n) + 24 5-7
336.19 (g)vaf(l) + 25 6-7 237.12 (v)af(l) + 26 6-10 615.36
(v)aflhk(k) + 27 6-10 615.36 (v)aflhk(k) + 28 6-12 857.50
(v)aflhkkn(d) + 29 6-12 857.50 (v)afhkkn(d) + 30 7-8 279.17
(a)fl(h) + 31 10-16 861.43 (h)kkndide( ) + 32 11-14 489.27
(k)kndi(d) + 33 12-15 476.2- (k)ndid(e) +
[0022] The malignin peptide was isolated from membranes of
glioblastoma multiforme (glioma) cells grown in tissue culture
(U.S. Pat. No. 6,242,578 B1). The sequence of a 16-mer peptide of
malignin was determined by hydrolysis and mass spectrometry: (SEQ
ID NO.: 4) ykagvaflhkkndide (Table 1). A search of published human
genome sequences for sequence encoding the 16-mer amino acid
sequence was negative. Since this 16-mer peptide was absent from
normal human genome data a search was made of sequences of other
organisms for possible origins and homologues. No identical
sequences were found. But, using the sequence of the 16-mer peptide
as a template, and constructing a "3-point-recognition" method to
visually scan protein sequences of several different organisms, a
new class of peptides, the replikins, was revealed in organisms as
diverse as algae, yeast and viruses. Surprisingly, these peptides
were found to be concentrated in larger `replicating` and
`transforming` proteins (so designated by their investigators,
based on activities, see Table 2).
[0023] Table 2 illustrates several replikin sequences that were
identified by the 3-point-recognition method of the invention.
TABLE-US-00002 TABLE 2 Examples of replikins in various organisms -
prototype: Glioma replikin* kagvaflhkk (SEQ ID No.: 1) SEQ ID NO.
Algae: 34 Caldophera prolifera kaskftkh 35 Isolepis prolifera
kaqaetgeikgh Yeast: 36 Schizosaccharomyces pombe ksfkypkkhk 37
Oryza sativa kkaygnelhk 2 Sacch. cerevisiae replication binding
protein hsikrelgiifdk Fungi: 38 Isocitrate lyase ICI l, Penicillium
marneffei kvdivthqk 39 DNA-dependent RNA polymerase 11, Diseula
dcstructiva kleedaayhrkk 40 Ophiostoma novo-ulm 1, RNA in Dutch elm
disease fungus kvilplrgnikgiffkh Amoeba: 41 Entamoeba invadens,
histone H2B klilkgdlnkh Bacteria: 42 Pribosomal protein replication
factor, Helicobacter pylori ksvhaflk Replication-associated protein
Staph. aureus 10 Mycoplasma pulmonic, chromosome replication
kkektthnk 43 Macrophage infectivity potentiator, L. legionella
kvhffqlkk 90 Bacillus anthracis kihlisvkk 91 Bacillus anthracis
hvkkekeknk 92 Bacillus anthracis khivkievk 93 Bacillus anthracis
kkkkikdiygkdallh 94 Bacillus anthracis kwekikqh 95 Bacillus
anthracis kklqipppiepkkddiih 96 Bacillus anthracis
hnryasnivesayllilnewkn- niqsdlikk 97 Bacillus anthracis
havddyagylldknqsdlvt- nskk 98 Bacillus anthracis haerlkvqknapk
Plants: 44 Arabidopsis thaliana, prolifera kdhdfdgdk 45 Arabidopsis
thaliana, cytoplasmic ribosomal kmkglkqkkah 46 Arabidopsis
thaliana, DNA binding protein kelssttqeksh Viruses: 9 Replication
associated protein A [Maize streak virus] kekkpskdeimrdiish 11
Bovine herpes virus 4, DNA replication protein hkinitngqk 12
Meleagrid herpesvirus 1, replication binding protein hkdlyrllmk 47
Feline immunodeficiency hlkdyklvk 3 Foot and Mouth Disease (O)
hkqkivapvk 5 HIV Type 1 kcfncgkegh 7 HIV Type 2 kcwncgkegh 99 Small
Pox Virus (Variola) khynnitwyk 100 Small Pox Virus (Variola)
kysqtgkeliih 101 Small Pox Virus (Variola) hyddvrikndivvsrck 102
Small Pox Virus (Variola) hrfklildsk 103 Small Pox Virus (Variola)
kerghnyyfek Tumor 48 Rous sarcoma virus tyrosine-protein kinase
kklrhek Viruses: 49 v-yes, avian sarcoma kklrhdk 50 c-yes, colon
cancer, malignant melanoma kklrhdk 51 v-srcC., avian sarcoma
kklrhek 52 c-src, colon, mammary, panrcreatic cancer kklrhek 53
Neuroblastoma RAS viral (v-ras) oncogene kqahelak 54 VPl (major
capsid protein) [Polyamavirus sp.] kthrfskh 55 Sindbis knlhekik 56
El [Human papilloamavirus type 71] khrpllqlk 57 v-erbB from AEV and
c-erb kspnhvk 58 v-fms (feline sarcoma) knihlekk 59 c-fms (acute
and chronic myelomonocytic tumors) knihlekk 60 large t-antigen I
[Polyomavirus sp. l kphlaqslek 61 middle t-antigen [Polyomavirus
sp, l- kqhrelkdk 62 small t-antigen [Polyomavirus spJ, kqhrelkdk 63
v-abl, murine acute leukemia kvpvlisptlkh 64 Human T-cell
lymphotropic virus typo 2 kslllevdkdish 65 c-kit, GI tumors, small
cell lung carcinoma kagitimvkreyh 18 Hepatitis C hyppkpgcivpak
Trans- 66 Transforming protein myb ksgkhlgk forming 67 Transforming
protein myc, Burkitt lymphoma krreqlkhk Proteins: 68 Ras-related
GTP-binding protein ksfevikvih 69 Transforming protein ras
(teratocarcinoma) kkkhtvkk 70 TRAF-associated NF kB activator TANK
kaqkdhlsk 71 RFP transforming protein hlkrvkdlkk 72 Transforming
protein D (S.C.) kygspkhrlik 73 Papilloma virus type 11,
transforming protein klkhilgkarfik 74 Protein tryosine kinasc (EC
2.7.1.ll2slk kgdhvkhykirk 75 Transforming protein (axl(-))
keklrdvmvdrhk 76 Transforming protein (N-myc) klqarqqqllkkieh 77
Fibroblast growth factor 4 (Kaposi sarcoma) kkgnrvsptmkvth Cancer
78 Matrix metaloproteinase 7 (uterine) keiplhfrk Cell 79
Transcription factor 7-like kkkphikk Proteins: 80 Breast cancer
antigen NY-BR-87 ktrhdplak 81 BRCA-1-Associated Ring Domain Protein
(breast) khhpkdnlik 82 `Autoantigen from a breast tumor` khkrkkfrqk
83 Glioma replikin (this study) kagvaflhkk 84 Ovarian cancer
antigen khkrkkfrqk 85 EE L leukemia kkkskkhkdk 86 Proto-oncogene
tyrosine-protein kinase C-ABLE hksekpalprk 87 Adenomatosis
polyposis coli kkkkpsrlkgdnek 88 Gastric cancer transforming
protein ktkkgnrvsptmkvth 89 Transforming protein (K-RAS 2B), lung
khkekmskdgkkkkkksk
[0024] Identification of an amino acid sequence as a replikin or as
containing a replikin, i.e., a homologue of the malignin 16-mer
peptide, requires that the three following "3-point recognition"
requirements be met. The peptide sequence must have (1) at least
one lysine residue located six to ten residues from another lysine
residue; (2) at least one histidine residue; and (3) a composition
of at least 6% lysine within an amino acid sequence of 7 to about
50 residues.
[0025] Databases were searched using the National Library of
Medicine keyword "PubMed" descriptor for protein sequences
containing replikin sequences. Sequences of all individual proteins
within each group of PubMed-classified proteins were visually
scanned for peptides meeting the three above-listed requirements.
An infrequent occurrence of homologues was observed in "virus
peptides" as a whole (1.5%), and in other peptides not designated
as associated with malignant transformation or replication such as
"brain peptides " and "neuropeptides" (together 8.5%).
Surprisingly, homologues were identified in 100% of "tumor
viruses", in 85% of "transforming proteins", and in 97% of "cancer
cell proteins" (FIG. 1). The peptides identified by this search
were named replikins, and a ten amino acid portion of the 16-mer
peptide, (SEQ ID NO.: 1) "kagvaflhkk", was named the glioma
replikin.
[0026] To permit classification of subtypes of replikins,
additional or "auxiliary specifications" to the basic
"3-point-recognition" requirements may be added: (a) on a
structural basis, such as the common occurrence of adjacent di- and
polylysines in cancer cell proteins (e.g., Transforming protein P21
B(K-RAS 2B), lung, Table 2, SEQ ID NO.: 89), and other adjacent
di-amino acids in TOLL-like receptors, or b) on a functional basis,
such as exhibiting ATPase, tyrosine kinase or redox activity as
seen in Table 2.
[0027] Whether replikin structures are conserved or are subject to
extensive natural mutation was examined by scanning the protein
sequences of various isolates of foot and mouth disease virus
(FMDV), where mutations in proteins of these viruses have been well
documented worldwide for decades. Protein sequences of FMDV
isolates were visually examined for the presence of both the entire
replikin and each of the component replikin amino acid residues
observed in a particular replikin. For example, in the protein VP1
of FMDV type O, the replikin (SEQ ID NO.: 3) "hkqkivapvk" was found
to be conserved in 78% of the 236 isolates reported in PubMed, and
each amino acid was found to be conserved in individual isolates as
follows: his, 95.6%; lys, 91.8%; gln 92.3%; lys, 84.1%; ile, 90.7%;
val, 91.8%; ala, 97.3%; pro, 96.2%; ala, 75.4%; and lys, 88.4%. The
high rate of conservation suggests structural and fimctional
stability of the replikin structure. Similarly, sequence
conservation was observed in different isolates of HIV for its
replikins, such as (SEQ ID NO.: 5) "kcfncgkegh" or (SEQ ID NO.: 6)
"kvylawvpahk" in HIV Type 1 and (SEQ ID NO.: 7) "kcwncgkegh" in HIV
Type 2 (Table 2). Other examples of conservation are seen in the
constant presence of malignin in successive generations, over 10
years of tissue culture of glioma cells, and by the constancy of
affinity of the glioma replikin for antimalignin antibody isolated
by immunoadsorption from 8,090 human sera from the U.S., U.K.,
Europe and Asia (e.g., FIG. 5 and U.S. Pat. No. 6,242,578 B 1).
[0028] As seen in FIG. 2, during anaerobic respiration when the
rate of cell replication is increased, malignin is enriched. That
is, malignin is found to increase not simply in proportion to the
increase in cell number and total membrane proteins, but is
enriched as much as tenfold in concentration, starting with 3% at
rest and reaching 30% of total membrane protein. This clear
demonstration of a marked increase in replikin concentration with
glioma cell replication points to and is consistent with the
presence of replikins here sought by the 3-point recognition method
and found in the proteins of various organisms which were found by
mutation studies and other previous studies to be critical to
replication. For example, replikins were identified in such
proteins as "Saccharomyces cerevisiae replication binding protein"
(SEQ ID NO.: 2) (hsikrelgiifdk); the "replication associated
protein A of maize streak virus" (SEQ ID NO.: 8) (kyivcareahk and
(SEQ ID NO.: 9) kekkpskdeimrdiish); the "replication-associated
protein of Staphylococcus aureus" (SEQ ID NO.: 10) (kkektthnk); the
"DNA replication protein of bovine herpes virus 4" (SEQ ID NO.: 11)
(hkinitngqk); and the "Mealigrid herpes virus 1 replication binding
protein" (SEQ ID NO.: 12) (hkdlyrllmk). Previous studies of tomato
leaf curl gemini virus show that the regulation of virus
accumulation appears to involve binding of amino acids 1-160 of the
"replicating protein" of that virus to leaf DNA and to other
replication protein molecules during virus replication. Analysis of
this sequence showed that amino acids 1-163 of this "replicating
protein" contain five replikins, namely: (SEQ ID NO.: 13)
kfrinaknyfltyph, (SEQ ID NO.: 14) knletpvnklfiricrefh, (SEQ ID NO.:
15) hpniqaaksstdvk, (SEQ ID NO.: 16) ksstdvkaymdkdgdvldh, and (SEQ
ID NO.: 17) kasalnilrekapkdfvlqfh.
[0029] Table 2 shows that replikin-containing proteins also are
associated frequently with redox functions, and protein synthesis
or elongation, as well as with cell replication. The association
with metal-based redox functions, the enrichment of the
replikin-containing glioma malignin concentration during anaerobic
replication, and the cytotoxicity of antimalignin at low
concentrations (picograms/cell) (FIG. 4c-f), all suggest that the
replikins are related to central respiratory functions, which are
perhaps less often subjected to the mutations characteristic of
proteins of more superficial location or less central survival
function.
[0030] Of particular interest were eight different replikins
identified in Bacillus anthracis, the organism responsible for
anthrax infections; and five different replikins identified in
small pox virus. The eight Bacillus anthracis peptides are present
in the Anthrax Toxin Lethal Factor Protein pX01-107 and have the
amino acid sequence of SEQ ID NO. 91, SEQ ID NO.92, SEQ ID NO. 93,
SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97 and SEQ
ID NO. 98, respectively. The five small pox virus peptides are
present in the Small Pox Virus Surface Antigen S Precursor Protein,
which purportedly enhances Small Pox Virus replication. The five
peptides have the amino acid sequence of SEQ ID NO. 99, SEQ ID NO.
100, SEQ ID NO. 101, SEQ ID NO. 102 and SEQ ID NO. 103,
respectively.
[0031] Data on anti-replikin antibodies support replikin class
unity. An anti-replikin antibody response has been quantified by
immunoadsorption of serum antimalignin antibody to immobilized
malignin (see Methods in U.S. Pat. No. 5,866,690). The abundant
production of antimalignin antibody by administration to rabbits of
the synthetic version of the 16-mer peptide whose sequence was
derived from malignin, absent carbohydrate or other groups, has
established rigorously that this peptide alone is an epitope, that
is, it is a sufficient basis for this immune response (FIG. 3). The
16-mer peptide produced both IgM and IgG forms of the antibody.
Antimalignin antibody was found to be increased in concentration in
serum in 37% of 79 cases in the U.S. and Asia of hepatitis B and C,
early, in the first five years of infection, long before the usual
observance of liver cancer, which develops about fifteen to
twenty-five years after infection. Relevant to both infectious
hepatitis and HIV infections, transformed cells may be one form of
safe haven for the virus: prolonging cell life and avoiding virus
eviction, so that the virus remains inaccessible to anti-viral
treatment.
[0032] A synthetic replikin vaccine such as the glioma replikin
(SEQ ID NO.: 1) "kagvaflhkk" or the hepatitis C replikin (SEQ ID
NO.: 18) "hyppkpgcivpak", or HIV replikins such as (SEQ ID NO.: 5)
"kcfncgkegh" or (SEQ ID NO.: 6) "kvylawvpahk" may be used to
augment antibody concentration in order to lyse the respective
virus infected cells and release virus extracellularly where
chemical treatment can then be effective. Recognin and/or replikin
peptides may be administered to a subject to induce the immune
system of the subject to produce anti-replikin and/or anti-recognin
antibodies. Generally, a 0.5 to about 2 mg dosage, preferably a 1
mg dosage of each peptide is administered to the subject to induce
an immune response. Subsequent dosages may be administered if
desired.
[0033] In another embodiment of the invention, isolated recognin or
replikin peptides may be used to generate antibodies. Various
procedures known in the art may be used for the production of
antibodies to replikin sequences or recognin sequences. Such
antibodies include but are not limited polyclonal, monoclonal,
chimeric, humanized, single chain, Fab fragments and fragments
produced by an Fab expression library. Antibodies that are linked
to a cytotoxic agent may also be generated.
[0034] For the production of antibodies various host animals may be
immunized by injection with a replikin or recognin peptide,
including but not limited to rabbits, mice, rats, and larger
mammals. Various adjuvants may be used to enhance the immunological
response, depending on the host species, including but not limited
to Freund's (complete and incomplete), mineral gels, such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, key limpet
hemocyanin, dintrophenol, and potentially useful human adjuvants
such as BCG and Corynebacterium parvum.
[0035] Monoclonal antibodies to replikins or recognins may be
prepared by using any technique that provides for the production of
antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally
described by Kohler and Milstein, (Nature, 1975, 256:495-497), the
human B-cell hybridoma technique (Kosbor et al., 1983, Immunology
Today, 4:72), and the EBV hybridoma technique (Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). In addition, techniques developed for the production of
chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci
USA, 81:6851-6855) may be used. Alternatively, techniques described
for the production of single chain antibodies (U.S. Pat. No.
4,946,778) can be adapted to produce replikin- or recognin-specific
single chain antibodies.
[0036] Particularly useful antibodies of the invention are those
that specifically bind to replikin sequences contained in peptides
and/or polypeptides of Bacillus anthracis. For example, antibodies
to any of peptides SEQ ID NO. 91, SEQ ID NO.92, SEQ ID NO. 93, SEQ
ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO.
98 and combinations of such antibodies are useful in the treatment
and/or prevention of anthrax.
[0037] Similarly, antibodies to peptides SEQ ID NO. 99, SEQ ID NO.
100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO.103 and combinations
of such antibodies are useful in the treatment and/or prevention of
small pox.
[0038] Antibody fragments which contain binding sites for a
replikin or recognin may be generated by known techniques. For
example, such fragments include but are not limited to F(ab').sub.2
fragments which can be produced by pepsin digestion of the antibody
molecules and the Fab fragments that can be generated by reducing
the disulfide bridges of the F(ab').sub.2 fragments. Alternatively,
Fab expression libraries can be generated (Huse et al., 1989,
Science, 246:1275-1281) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity.
[0039] The fact that antimalignin antibody is increased in
concentration in human malignancy regardless of cancer cell type
(FIG. 5), and that this antibody binds to malignant cells
regardless of cell type now may be explained by the presence of the
replikin structures present in most malignancies (FIG. 1 and Table
2). Population studies have shown that antimalignin antibody
increases in concentration in healthy adults with age, and more so
in high-risk families, as the frequency of cancer increases. An
additional two-fold or greater antibody increase which occurs in
early malignancy has been independently confirmed with a
sensitivity of 97% in breast cancers 1-10 mm in size. Shown to
localize preferentially in malignant cells in vivo, histochemically
the antibody does not bind to normal cells but selectively binds to
(FIGS. 4a,b) and is highly cytotoxic to transformed cells in vitro
(FIG. 4c-f). Since in these examples the same antibody is bound by
several cell types, that is, brain glioma, hematopoietic cells
(leukemia), and small cell carcinoma of lung, malignant replikin
class unity is again supported.
[0040] Antimalignin does not increase with benign proliferation,
but specifically increases only with malignant transformation and
replication in breast in vivo and returns from elevated to normal
values upon elimination of malignant cells (FIG. 5). Antimalignin
antibody concentration has been shown to relate quantitatively to
the survival of cancer patients, that is, the more antibody, the
longer the survival. Taken together, these results suggest that
antireplikin antibodies may be a part of a mechanism of control of
cell transformation and replication. Augmentation of this immune
response may be useful in the control of replication, either
actively with synthetic replikins as vaccines, or passively by the
administration of anti-replikin antibodies, or by the introduction
of non-immune based organic agents, such as for example,
carbohydrates, lipids and the like, which are similarly designed to
target the replikin specifically. For organisms such as diatom
plankton, foot and mouth disease virus, tomato leaf curl gemini
virus, hepatitis B and C, and HIV, and malignant cells, identified
constituent replikins are useful as vaccines, and also may be
usefully targeted for diagnostic purposes.
[0041] The replikin sequence structure is associated with the
function of replication. Thus, whether the replikins of this
invention are used for targeting sequences that contain replikins
for the purpose of diagnostic identification, promoting
replication, or inhibiting or attacking replication, for example,
the structure-function relationship of the replikin is fundamental.
Thus, while the structure of the replikin may be a part of a larger
protein sequence, which may have been previously identified, it is
necessary to utilize only the specific replikin structure when
seeking to induce antibodies that will recognize and attach to the
replikin fragment and thereby cause destruction of the cell. Even
though the larger protein sequence may be known in the art as
having a "replication associated function," vaccines using the
larger protein often have failed or proven ineffective, even though
they contain one or more replikin sequences.
[0042] Although the present inventors do not wish to be held to a
single theory, the studies herein suggest that the prior art
vaccines are ineffective because they are based on the use of the
larger protein sequence. The larger protein sequence invariably has
one or more epitopes (independent antigenic sequences that can
induce specific antibody formation); replikin structures usually
comprise one of these potential epitopes. The presence of other
epitopes within the larger protein may interfere with adequate
formation of antibodies to the replikin, See, e.g., Webster, R. G.,
J. Immunol., 97(2):177-183 (1966); and Webster et al., J. Infect.
Dis., 134:48-58, 1976; Klenerman et al, Nature 394:421-422 (1998)
for a discussion of the well-known phenomenon "original antigenic
sin"). The formation of an antibody to a non-replikin epitope may
allow binding to the cell, but not necessarily lead to cell
destruction.
[0043] It is well known in the art that in the course of antibody
production against a "foreign" protein, the protein is first
hydrolyzed into smaller fragments. Usually fragments containing
from about six to ten amino acids are selected for antibody
formation. Thus, if hydrolysis of a protein does not result in
replikin-containing fragments, anti-replikin antibodies will not be
produced. In this regard, it is interesting that replikins contain
lysine residues located six to ten amino acids apart, since lysine
residues are known to bind to membranes.
[0044] Furthermore, replikin sequences contain at least one
histidine residue. Histidine is frequently involved in binding to
redox centers. Thus, an antibody that specifically recognizes a
replikin sequence has a better chance of inactivating or destroying
the cell in which the replikin is located, as seen with
anti-malignin antibody, which is perhaps the most cytotoxic
antibody yet described, being active at picograms per cell.
[0045] One of the reasons that vaccines directed towards a
particular protein antigen of a disease causing agent have not been
fully effective in providing protection against the disease (such
as foot and mouth vaccine which has been developed against the VP1
protein or large segments of the VP1 protein) is that antibody to
the replikins have not been produced. That is, either epitopes
other than replikins present in the larger protein fragments may
interfere according to the phenomenon of "original antigenic sin",
and/or because the hydrolysis of larger protein sequences into
smaller sequences for processing to produce antibodies results in
loss of integrity of any replikin structure that is present, e.g.,
the replikin is cut in two and/or the histidine residue is lost in
the hydrolytic processing. The present studies suggest that for an
effective vaccine to be produced, the replikin sequences, and no
other epitope, should be used as the vaccine. For example, a
vaccine of the invention can be generated using any one of the
replikin peptides identified by the three point recognition system.
Particularly preferred peptides for an anthrax vaccine include
peptides SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO.
94, SEQ ID NO.95, SEQ ID NO. 96, SEQ ID NO.97, SEQ ID NO.98, and
combinations thereof. Preferred peptides for use as a small pox
vaccine are peptides SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101,
SEQ ID NO. 102, SEQ ID NO. 103 and combinations thereof. These
peptides, alone or in various combinations are administered to a
subject, preferably by i.v. or intramuscular injection, in order to
stimulate the immune system of the subject to produce antibodies to
the peptide. Generally the dosage of peptides is in the range of
from about 0.1 .mu.g to about 10 mg, preferably about 10 .mu.g to
about 1 mg, and most preferably about 50 .mu.g to about 500 .mu.g.
The skilled practitioner can readily determine the dosage and
number of dosages needed to produce an effective immune
response.
[0046] Replikin or recognin DNA or RNA may have a number of uses
for the diagnosis of diseases resulting from infection with a
virus, bacterium or other replikin or recognin encoding agent. For
example, replikin or recognin nucleotide sequences may be used in
hybridization assays of biopsied tissue to diagnose the presence of
a particular organism, e.g., Southern or Northern analysis,
including in situ hybridization assays.
[0047] Also within the scope of the invention are
oligoribonucleotide sequences, that include antisense RNA and DNA
molecules and ribozymes that function to inhibit the translation of
replikin- or recognin-containing mRNA. Both antisense RNA and DNA
molecules and ribozymes may be prepared by any method known in the
art. The antisense molecules can be incorporated into a wide
variety of vectors for delivery to a subject. The skilled
practitioner can readily determine the best route of delivery,
although generally i.v. or i.m. delivery is routine. The dosage
amount is also readily ascertainable.
[0048] Particularly preferred antisense nucleic acid molecules are
those that are complementary to a mRNA encoding a Bacillus
anthracis polypeptide comprising a replikin sequence comprising
from 7 to about 50 amino acids including (1) at least one lysine
residue located six to ten residues from a second lysine residue;
(2) at least one histidine residue; and (3) at least 6% lysine
residues. More preferred are antisense nucleic acid molecules that
are complementary to the coding strand of the gene or to the mRNA
encoding the Bacillus anthracis Anthrax Lethal Factor Protein
pX01-107 peptide, wherein the antisense nucleic acid molecule is
complementary to a nucleotide sequence encoding the peptide of SEQ
ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO.
95, SEQ ID NO. 96, SEQ ID NO. 97, or SEQ ID NO. 98.
[0049] Another preferred set of antisense nucleic acid molecules
includes those that are complementary to a mRNA encoding a Small
Pox Virus polypeptide comprising a replikin sequence comprising
from 7 to about 50 amino acids including (1) at least one lysine
residue located six to ten residues from a second lysine residue;
(2) at least one histidine residue; and (3) at least 6% lysine
residues. More preferred are antisense nucleic acid molecules that
are complementary to the coding strand of the gene or to the mRNA
encoding the Small Pox Virus Surface Antigen S Precursor Protein,
wherein the antisense nucleic acid molecule is complementary to a
nucleotide sequence encoding the peptide of SEQ ID NO. 99, SEQ ID
NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, or SEQ ID NO. 103.
[0050] In another embodiment of the invention, immune serum
containing antibodies to one or more replikin obtained from an
individual exposed to one or more replikins may be used to induce
passive immunity in another individual or animal. Immune serum may
be administered via i.v. to a subject in need of treatment. Passive
immunity also can be achieved by injecting a recipient with
preformed antibodies to one or more replikins. Passive immunization
may be used to provide immediate protection to individuals who have
been exposed to an infectious organism. Administration of immune
serum or preformed antibodies is routine and the skilled
practitioner can readily ascertain the amount of serum or
antibodies needed to achieve the desired effect.
[0051] Visual scanning of over three thousand sequences was
performed in developing the present 3-point-recognition methods.
However, data banks comprising nucleotide and/or amino acid
sequences can also be scanned by computer for the presence of
sequences meeting the 3 point recognition requirements.
[0052] The three point recognition method may also be modified to
identify other useful compounds of covalently linked organic
molecules, including other covalently linked amino acids,
nucleotides, carbohydrates, lipids or combinations thereof. In this
embodiment of the invention a sequence is screened for subsequences
containing three or more desired structural characteristics. In the
case of screening compounds composed of covalently linked amino
acids, lipids or carbohydrates the subsequence of 7 to about 50
covalently linked units should contain (1) at least one first amino
acid, carbohydrate or lipid residue located six to ten residues
from a second of the first amino acid, carbohydrate or lipid
residue; (2) at least one second amino acid, lipid or carbohydrate
residue; and (3) at least 6% of the first amino acid, carbohydrate
or lipid residue. In the case of screening nucleotide sequences,
the subsequence of about 21 to about 150 nucleotides should contain
(1) at least one first amino acid residue located within eighteen
to thirty nucleotides from a second codon encoding the first amino
acid residue; (2) at least one second amino acid residue; and (3)
encodes at least 6% of said first amino acid residue.
[0053] According to another embodiment of the invention, the
methods described herein may be performed by a computer. FIG. 6 is
a block diagram of a computer available for use with the foregoing
embodiments of the present invention. The computer may include a
processor, an input/output device and a memory storing executable
program instructions representing the 3-point-recognition methods
of the foregoing embodiments. The memory may include a static
memory, volatile memory and/or a nonvolatile memory. The static
memory conventionally may be a read only memory ("ROM") provided on
a magnetic, or an electrical or optical storage medium. The
volatile memory conventionally may be a random access memory
("RAM") and may be integrated as a cache within the processor or
provided externally from the processor as a separate integrated
circuit. The non-volatile memory may be an electrical, magnetic or
optical storage medium.
EXAMPLE 1
Process for Extraction, Isolation and Identification of Replikins
and the Use of Replikins to Target, Label or Destroy
Replikin-Containing Organisms
[0054] a) Algae
[0055] The following algae were collected from Bermuda water sites
and either extracted on the same day or frozen at -20 degrees C.
and extracted the next day. The algae were homogenized in a cold
room (at 0 to 5 degrees C.) in 1 gram aliquots in neutral buffer,
for example 100 cc. of 0.005M phosphate buffer solution, pH7
("phosphate buffer") for 15 minutes in a Waring blender,
centrifuged at 3000 rpm, and the supernatant concentrated by
perevaporation and dialyzed against phosphate buffer in the cold to
produce a volume of approximately 15 ml. The volume of this extract
solution was noted and an aliquot taken for protein analysis, and
the remainder was fractionated to obtain the protein fraction
having a pK range between 1 and 4. The preferred method of
fractionation is chromatography as follows:
[0056] The extract solution is fractionated in the cold room (4
degrees C.) on a DEAE cellulose (Cellex-D) column 2.5.times.11.0
cm, which has been equilibrated with 0.005M phosphate buffer.
Stepwise eluting solvent changes are made with the following
solutions:
[0057] Solution 1--4.04 g. NaH2P04 and 0.5 g NaH2P04 are dissolved
in 15 litres of distilled water (0.005 molar, pH7);
[0058] Solution 2--8.57 g. NaH2P04 is dissolved in 2,480 ml. of
distilled water;
[0059] Solution 3--17.1 g. of NaH2P04 is dissolved in 2480 ml of
distilled water (0.05 molar, pH 4.7);
[0060] Solution 4--59.65 g. of NaH2P04 is dissolved in 2470 ml
distilled water (0.175 molar);
[0061] Solution 5--101.6 g. of NaH2P04 is dissolved in 2455 ml
distilled water (pH 4.3);
[0062] Solution 6--340.2 g. of NaH2P04 is dissolved in 2465 of
distilled water (1.0 molar, pX-i 4.1);
[0063] Solution 7--283.63 g. of 80% phosphoric acid (H3P04) is made
up in 2460 ml of distilled water (1.0 molar, pH 1.0).
[0064] The extract solution, in 6 to 10 ml volume, is passed onto
the column and overlayed with Solution 1, and a reservoir of 300 ml
of Solution 1 is attached and allowed to drip by gravity onto the
column. Three ml aliquots of eluant are collected and analyzed for
protein content at OD 280 until all of the protein to be removed
with Solution 1 has been removed from the column. Solution 2 is
then applied to the column, followed in succession by Solutions 3,
4, 5, 6 aid 7 until all of the protein which can, be removed with
each Solution is removed from the column. The eluates from Solution
7 are combined, dialyzed against phosphate buffer, the protein
content determined of both dialysand and dialyzate, and both
analyzed by gel electrophoresis. One or two bands of peptide or
protein of molecular weight between 3,000 and 25,000 Daltons are
obtained in Solution 7. For example the algae Caulerpa mexicana,
Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpa
verticillata, Halimeda tuna, and Penicillos capitatus, after
extraction and treatment as above, all demonstrated in Solution 7
eluates sharp peptide bands in this molecular weight region with no
contaminants. These Solution 7 proteins or their eluted bands are
hydrolyzed, and the amino acid composition determined. The peptides
so obtained, which have a lysine composition of 6% or greater are
Replikin precursors. These Replikin peptide precursors are then
determined for amino acid sequence by hydrolysis and mass
spectrometry as detailed in U.S. Pat. No. 6,242,578 B1. Those which
fulfill the criteria defined by the "3-point-recognition" method
are identified as Replikins. This procedure can also be applied to
obtain yeast, bacterial and any plant Replikins.
[0065] b) Virus
[0066] Using the same extraction and column chromatography
separation methods as above in a) for algae, Replikens in
virus-infected cells are isolated and identified.
[0067] c) Tumor Cells in vivo and in vitro Tissue Culture
[0068] Using the same extraction and column chromatography
separation methods as above in a) for algae, Replikins in tumor
cells are isolated and identified. For example, Replikin precursors
of Astrocytin isolated from malignant brain tumors, Malignin
(Aglyco 1OB) isolated from glioblastoma tumor cells in tissue
culture, MCF7 mammary carcinoma cells in tissue culture, and
P.sub.3J Lymphoma cells in tissue culture each treated as above in
a) yielded Replikin precursors with lysine content of 9.1%, 6.7%,
6.7%, and 6.5% respectively. Hydrolysis and mass spectrometry of
Aglyco 1OB as described in Example 10 U.S. Pat. No. 6,242,578 B1
produced the amino acid sequence, ykagvaflhkkndiide the 16-mer
Replikin.
EXAMPLE 2
[0069] As an example of diagnostic use of Replikins: Aglyco 1OB or
the 16-mer Repliken may be used as antigen to capture and quantify
the amount of its corresponding antibody present in serum for
diagnostic purposes are as shown in FIGS. 2, 3, 4 and 7 of U.S.
Pat. No. 6,242,578 B1.
[0070] As an example of the production of agents to attach to
Replikins for labeling, nutritional or destructive purposes:
Injection of the 16-mer Replikin into rabbits to produce the
specific antibody to the 16-mer Replikin is shown in Example 6 and
FIGS. 9A and 9B of U.S. Pat. No. 6,242,578 B1.
[0071] As an example of the use of agents to label Replikins: The
use of antibodies to the 16-mer Replikin to label specific cells
which contain this Replikin is shown in FIG. 5 and Example 6 of
U.S. Pat. No. 6,242,578 B1.
[0072] As an example of the use of agents to destroy Replikins: The
use of antibodies to the 16-mer Replikin to inhibit or destroy
specific cells which contain this Replikin is shown in FIG. 6 of
U.S. Pat. No. 6,242,578 B1.
[0073] From a proteomic point of view the construction of a
"3-point recognition" template based on the new glioma peptide
sequence led directly to identification of a biology-wide class of
proteins having related structures and functions. The operation of
the 3-point-recognition method resembles identification by the use
of a "keyword" search; but instead of using the exact spelling of
the keyword "kagvafihkk" as in a typical sequence homology search,
or in the nucleotide specification of an amino acid, an abstraction
of the keyword delimited by the "3-point-recognition" parameters is
used. This delimited abstraction, although derived from a single
relatively short amino acid sequence leads to identification of a
class of proteins with structures that are defined by the same
specifications. That particular functions, in this case
transformation and replication, in addition to structures, turn out
also to be shared by members of the exposed class suggests that
these structures and functions are related. Thus, from this newly
identified short peptide sequence, a molecular recognition
`language` has been formulated, which previously has not been
described. Further, the sharing of immunological specificity by
diverse members of the class, as here demonstrated for the cancer
replikins, suggests that B cells and their product antibodies
recognize replikins by means of a similar recognition language.
Since "3-point-recognition" is a proteomic method that specifies a
particular class of proteins, using three or more different
recognition points for other peptides similarly should provide
useful information concerning other proteins classes. Further, the
"3-point-recognition" method is applicable to other recognins, for
example to the TOLL `innate` recognition of lipopolyssacharides of
organisms.
[0074] Several embodiments of the present invention are
specifically illustrated and described herein. However, it will be
appreciated that modifications and variations of the present
invention are encompassed by the above teachings and within the
purview of the appended claims without departing from the spirit
and intended scope of the invention.
Sequence CWU 1
1
103 1 10 PRT Artificial Sequence Description of Artificial Sequence
Synthetic glioma replikin 1 Lys Ala Gly Val Ala Phe Leu His Lys Lys
1 5 10 2 13 PRT Saccharomyces cerevisiae 2 His Ser Ile Lys Arg Glu
Leu Gly Ile Ile Phe Asp Lys 1 5 10 3 10 PRT Gemini vinis virus 3
His Lys Gln Lys Ile Val Ala Pro Val Lys 1 5 10 4 16 PRT Unknown
Organism Description of Unknown Organism Virus recognin 4 Tyr Lys
Ala Gly Val Ala Phe Leu His Lys Lys Asn Asp Ile Asp Glu 1 5 10 15 5
10 PRT Human immunodeficiency virus type 1 5 Lys Cys Phe Asn Cys
Gly Lys Glu Gly His 1 5 10 6 11 PRT Human immunodeficiency virus
type 1 6 Lys Val Tyr Leu Ala Trp Val Pro Ala His Lys 1 5 10 7 10
PRT Human immunodeficiency virus type 2 7 Lys Cys Trp Asn Cys Gly
Lys Glu Gly His 1 5 10 8 11 PRT Maize streak virus 8 Lys Tyr Ile
Val Cys Ala Arg Glu Ala His Lys 1 5 10 9 17 PRT Maize streak virus
9 Lys Glu Lys Lys Pro Ser Lys Asp Glu Ile Met Arg Asp Ile Ile Ser 1
5 10 15 His 10 9 PRT Staphylococcus aureus 10 Lys Lys Glu Lys Thr
Thr His Asn Lys 1 5 11 10 PRT Bovine herpesvirus 4 11 His Lys Ile
Asn Ile Thr Asn Gly Gln Lys 1 5 10 12 10 PRT Meleagrid herpesvirus
1 12 His Lys Asp Leu Tyr Arg Leu Leu Met Lys 1 5 10 13 15 PRT
Unknown Organism Description of Unknown Organism Virus recognin 13
Lys Phe Arg Ile Asn Ala Lys Asn Tyr Phe Leu Thr Tyr Pro His 1 5 10
15 14 19 PRT Unknown Organism Description of Unknown Organism Virus
recognin 14 Lys Asn Leu Glu Thr Pro Val Asn Lys Leu Phe Ile Arg Ile
Cys Arg 1 5 10 15 Glu Phe His 15 14 PRT Unknown Organism
Description of Unknown Organism Virus recognin 15 His Pro Asn Ile
Gln Ala Ala Lys Ser Ser Thr Asp Val Lys 1 5 10 16 19 PRT Unknown
Organism Description of Unknown Organism Virus recognin 16 Lys Ser
Ser Thr Asp Val Lys Ala Tyr Met Asp Lys Asp Gly Asp Val 1 5 10 15
Leu Asp His 17 21 PRT Unknown Organism Description of Unknown
Organism Virus recognin 17 Lys Ala Ser Ala Leu Asn Ile Leu Arg Glu
Lys Ala Pro Lys Asp Phe 1 5 10 15 Val Leu Gln Phe His 20 18 13 PRT
Hepatitis C virus 18 His Tyr Pro Pro Lys Pro Gly Cys Ile Val Pro
Ala Lys 1 5 10 19 4 PRT Homo sapiens 19 Tyr Lys Ala Gly 1 20 6 PRT
Homo sapiens 20 Tyr Lys Ala Gly Val Ala 1 5 21 7 PRT Homo sapiens
21 Tyr Lys Ala Gly Val Ala Phe 1 5 22 7 PRT Homo sapiens 22 Tyr Lys
Ala Gly Val Ala Phe 1 5 23 9 PRT Homo sapiens 23 Ala Gly Val Ala
Phe His Lys Lys Asn 1 5 24 4 PRT Homo sapiens 24 Gly Val Ala Phe 1
25 3 PRT Homo sapiens 25 Val Ala Phe 1 26 7 PRT Homo sapiens 26 Val
Ala Phe Leu His Lys Lys 1 5 27 7 PRT Homo sapiens 27 Val Ala Phe
Leu His Lys Lys 1 5 28 9 PRT Homo sapiens 28 Val Ala Phe Leu His
Lys Lys Asn Asp 1 5 29 8 PRT Homo sapiens 29 Val Ala Phe His Lys
Lys Asn Asp 1 5 30 4 PRT Homo sapiens 30 Ala Phe Leu His 1 31 8 PRT
Homo sapiens 31 His Lys Lys Asn Asp Ile Asp Glu 1 5 32 6 PRT Homo
sapiens 32 Lys Lys Asn Asp Ile Asp 1 5 33 6 PRT Homo sapiens 33 Lys
Asn Asp Ile Asp Glu 1 5 34 8 PRT Caldophera prolifera 34 Lys Ala
Ser Lys Phe Thr Lys His 1 5 35 12 PRT Isolepis prolifera 35 Lys Ala
Gln Ala Glu Thr Gly Glu Ile Lys Gly His 1 5 10 36 10 PRT
Schizosaccharomyces pombe 36 Lys Ser Phe Lys Tyr Pro Lys Lys His
Lys 1 5 10 37 10 PRT Oryza sativa 37 Lys Lys Ala Tyr Gly Asn Glu
Leu His Lys 1 5 10 38 9 PRT Penicillium marneffei 38 Lys Val Asp
Ile Val Thr His Gln Lys 1 5 39 12 PRT Diseula dcstructiva 39 Lys
Leu Glu Glu Asp Ala Ala Tyr His Arg Lys Lys 1 5 10 40 17 PRT
Ophiostoma novo-ulmi 40 Lys Val Ile Leu Pro Leu Arg Gly Asn Ile Lys
Gly Ile Phe Phe Lys 1 5 10 15 His 41 11 PRT Entamoeba invadens 41
Lys Leu Ile Leu Lys Gly Asp Leu Asn Lys His 1 5 10 42 8 PRT
Helicobacter pylori 42 Lys Ser Val His Ala Phe Leu Lys 1 5 43 9 PRT
Mycoplasma pulmonis 43 Lys Val His Phe Phe Gln Leu Lys Lys 1 5 44 9
PRT Arabidopsis thaliana 44 Lys Asp His Asp Phe Asp Gly Asp Lys 1 5
45 11 PRT Arabidopsis thaliana 45 Lys Met Lys Gly Leu Lys Gln Lys
Lys Ala His 1 5 10 46 12 PRT Arabidopsis thaliana 46 Lys Glu Leu
Ser Ser Thr Thr Gln Glu Lys Ser His 1 5 10 47 9 PRT Feline
immunodeficiency virus 47 His Leu Lys Asp Tyr Lys Leu Val Lys 1 5
48 7 PRT Rous sarcoma virus 48 Lys Lys Leu Arg His Glu Lys 1 5 49 7
PRT Avian sarcoma virus 49 Lys Lys Leu Arg His Asp Lys 1 5 50 7 PRT
Homo sapiens 50 Lys Lys Leu Arg His Asp Lys 1 5 51 7 PRT Avian
sarcoma virus 51 Lys Lys Leu Arg His Glu Lys 1 5 52 7 PRT Homo
sapiens 52 Lys Lys Leu Arg His Glu Lys 1 5 53 8 PRT Homo sapiens 53
Lys Gln Ala His Glu Leu Ala Lys 1 5 54 8 PRT Polyama virus 54 Lys
Thr His Arg Phe Ser Lys His 1 5 55 8 PRT Sindbis virus 55 Lys Asn
Leu His Glu Lys Ile Lys 1 5 56 9 PRT Human papilloamavirus type 71
56 Lys His Arg Pro Leu Leu Gln Leu Lys 1 5 57 7 PRT Avian
encephalomyelitis virus 57 Lys Ser Pro Asn His Val Lys 1 5 58 8 PRT
Feline sarcoma virus 58 Lys Asn Ile His Leu Glu Lys Lys 1 5 59 8
PRT Homo sapiens 59 Lys Asn Ile His Leu Glu Lys Lys 1 5 60 10 PRT
Polyoma virus 60 Lys Pro His Leu Ala Gln Ser Leu Glu Lys 1 5 10 61
9 PRT Polyoma virus 61 Lys Gln His Arg Glu Leu Lys Asp Lys 1 5 62 9
PRT Polyoma virus 62 Lys Gln His Arg Glu Leu Lys Asp Lys 1 5 63 12
PRT Murine leukemia virus 63 Lys Val Pro Val Leu Ile Ser Pro Thr
Leu Lys His 1 5 10 64 13 PRT Human T-cell lymphotropic virus type 2
64 Lys Ser Leu Leu Leu Glu Val Asp Lys Asp Ile Ser His 1 5 10 65 13
PRT Homo sapiens 65 Lys Ala Gly Ile Thr Ile Met Val Lys Arg Glu Tyr
His 1 5 10 66 8 PRT Homo sapiens 66 Lys Ser Gly Lys His Leu Gly Lys
1 5 67 9 PRT Homo sapiens 67 Lys Arg Arg Glu Gln Leu Lys His Lys 1
5 68 10 PRT Homo sapiens 68 Lys Ser Phe Glu Val Ile Lys Val Ile His
1 5 10 69 8 PRT Homo sapiens 69 Lys Lys Lys His Thr Val Lys Lys 1 5
70 9 PRT Homo sapiens 70 Lys Ala Gln Lys Asp His Leu Ser Lys 1 5 71
10 PRT Homo sapiens 71 His Leu Lys Arg Val Lys Asp Leu Lys Lys 1 5
10 72 11 PRT Homo sapiens 72 Lys Tyr Gly Ser Pro Lys His Arg Leu
Ile Lys 1 5 10 73 13 PRT Papilloma virus type 11 73 Lys Leu Lys His
Ile Leu Gly Lys Ala Arg Phe Ile Lys 1 5 10 74 12 PRT Homo sapiens
74 Lys Gly Asp His Val Lys His Tyr Lys Ile Arg Lys 1 5 10 75 13 PRT
Homo sapiens 75 Lys Glu Lys Leu Arg Asp Val Met Val Asp Arg His Lys
1 5 10 76 15 PRT Homo sapiens 76 Lys Leu Gln Ala Arg Gln Gln Gln
Leu Leu Lys Lys Ile Glu His 1 5 10 15 77 14 PRT Homo sapiens 77 Lys
Lys Gly Asn Arg Val Ser Pro Thr Met Lys Val Thr His 1 5 10 78 9 PRT
Homo sapiens 78 Lys Glu Ile Pro Leu His Phe Arg Lys 1 5 79 8 PRT
Homo sapiens 79 Lys Lys Lys Pro His Ile Lys Lys 1 5 80 9 PRT Homo
sapiens 80 Lys Thr Arg His Asp Pro Leu Ala Lys 1 5 81 10 PRT Homo
sapiens 81 Lys His His Pro Lys Asp Asn Leu Ile Lys 1 5 10 82 10 PRT
Homo sapiens 82 Lys His Lys Arg Lys Lys Phe Arg Gln Lys 1 5 10 83
10 PRT Homo sapiens 83 Lys Ala Gly Val Ala Phe Leu His Lys Lys 1 5
10 84 10 PRT Homo sapiens 84 Lys His Lys Arg Lys Lys Phe Arg Gln
Lys 1 5 10 85 10 PRT Homo sapiens 85 Lys Lys Lys Ser Lys Lys His
Lys Asp Lys 1 5 10 86 11 PRT Homo sapiens 86 His Lys Ser Glu Lys
Pro Ala Leu Pro Arg Lys 1 5 10 87 14 PRT Homo sapiens 87 Lys Lys
Lys Lys Pro Ser Arg Leu Lys Gly Asp Asn Glu Lys 1 5 10 88 16 PRT
Homo sapiens 88 Lys Thr Lys Lys Gly Asn Arg Val Ser Pro Thr Met Lys
Val Thr His 1 5 10 15 89 18 PRT Homo sapiens 89 Lys His Lys Glu Lys
Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys 1 5 10 15 Ser Lys 90 9
PRT Legionella sp. 90 Lys Ile His Leu Ile Ser Val Lys Lys 1 5 91 10
PRT Bacillus anthracis 91 His Val Lys Lys Glu Lys Glu Lys Asn Lys 1
5 10 92 9 PRT Bacillus anthracis 92 Lys His Ile Val Lys Ile Glu Val
Lys 1 5 93 16 PRT Bacillus anthracis 93 Lys Lys Lys Lys Ile Lys Asp
Ile Tyr Gly Lys Asp Ala Leu Leu His 1 5 10 15 94 8 PRT Bacillus
anthracis 94 Lys Trp Glu Lys Ile Lys Gln His 1 5 95 18 PRT Bacillus
anthracis 95 Lys Lys Leu Gln Ile Pro Pro Pro Ile Glu Pro Lys Lys
Asp Asp Ile 1 5 10 15 Ile His 96 31 PRT Bacillus anthracis 96 His
Asn Arg Tyr Ala Ser Asn Ile Val Glu Ser Ala Tyr Leu Leu Ile 1 5 10
15 Leu Asn Glu Trp Lys Asn Asn Ile Gln Ser Asp Leu Ile Lys Lys 20
25 30 97 24 PRT Bacillus anthracis 97 His Ala Val Asp Asp Tyr Ala
Gly Tyr Leu Leu Asp Lys Asn Gln Ser 1 5 10 15 Asp Leu Val Thr Asn
Ser Lys Lys 20 98 13 PRT Bacillus anthracis 98 His Ala Glu Arg Leu
Lys Val Gln Lys Asn Ala Pro Lys 1 5 10 99 10 PRT Variola virus 99
Lys His Tyr Asn Asn Ile Thr Trp Tyr Lys 1 5 10 100 12 PRT Variola
virus 100 Lys Tyr Ser Gln Thr Gly Lys Glu Leu Ile Ile His 1 5 10
101 17 PRT Variola virus 101 His Tyr Asp Asp Val Arg Ile Lys Asn
Asp Ile Val Val Ser Arg Cys 1 5 10 15 Lys 102 10 PRT Variola virus
102 His Arg Phe Lys Leu Ile Leu Asp Ser Lys 1 5 10 103 11 PRT
Variola virus 103 Lys Glu Arg Gly His Asn Tyr Tyr Phe Glu Lys 1 5
10
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